Molecular and functional characterization of olfaction genes in tsetse flies

描述(由申请人提供)： 这项研究的总体目标是提供舌叶载体中调节其嗅觉过程的分子过程的信息，使它们能够定位它们的宿主和其他对它们的生存和繁殖至关重要的资源。这些目标将通过在肯尼亚选定的具有医疗和兽医重要性的采采树物种(苍白舌蝇、澳大利亚舌蝇和绒毛舌蝇)中寻找和验证嗅觉反应基因来实现。我们提出了一种综合的跨学科的方法来：1)确定肯尼亚Mbita的Shimba丘陵和Glossina fuscipes的共域Glossina pallidipe和Glossina auustin的嗅觉反应基因的差异；2)确定肯尼亚Mbita地区的Shimba丘陵和G.F.fuscipes的共域Glossina pallidipe和G.Austi的嗅觉反应基因的功能作用。我们假设这些果蝇之间的嗅觉基因是同源的，差异表达，并显著影响果蝇对不同气味的反应。将从各自的采采点采集选定采采种的成虫样本，并从头部组织中提取RNA。我们将开展一个发现项目，通过使用下一代测序技术来确定不同物种之间表现出的全球转录组水平的异质性。从苍蝇肠道内容物中提取的DNA将进行带有COI标记的PCR扩增，以从生命数据系统条形码(BLOD)数据库中确定可能的宿主物种。在不同物种的比较转录组分析之后，我们将通过qPCR在体外验证选定的一组可能的嗅觉相关基因的存在和表达谱。选定的嗅觉基因的功能作用(S)将通过RNA干扰基因沉默方法结合电子天线图和风洞技术进一步研究，以确定这些基因在调节苍蝇生理和行为方面的作用。有关嗅觉相关基因及其各自在调节采采蝇嗅觉行为中的作用的全面信息的获得，将为进一步开发利用这些基因来控制采采蝇媒介奠定基础。这项研究将主要在肯尼亚的国际昆虫中心进行 生理学、生态学和肯尼亚农业研究所--锥虫病研究中心与Paul Mireji合作，附带赠款为全球传染病D43 TW007391(08/01/2008-03/31/2013)。

DESCRIPTION (provided by applicant): The overall goal of this study is to provide information on molecular processes in tsetse vectors that mediate their olfactory processes, enabling them to locate their hosts and other critical resources for their survival and reproduction. The goals will be achieved by prospecting for and validating olfaction responsive genes in selected tsetse species (Glossina pallidipes, Glossina austeni and Glossina fuscipes fuscipes) of medical and veterinary importance in Kenya. We propose an integrated and interdisciplinary approach to: 1) Determine differences in olfactory responsive genes in sympatric Glossina pallidipes and Glossina austeni in Shimba hills and Glossina fuscipes fuscipes in Mbita in Kenya and 2) Determine functional roles of olfactory responsive genes in sympatric G. pallidipes and G. austeni in Shimba hills and G. f. fuscipes in Mbita districts in Kenya. We hypothesize that putative olfactory genes among these flies are orthologuous, differentially expressed and significantly influence responses of the flies to different odors. Adults of the selected tsetse species will be sampled from respective sites and RNA will be extracted from head tissues. We will undertake a discovery project by using next generation sequencing technologies to determine global transcriptome-level heterogeneities exhibited between the different species. The DNA obtained from the gut contents of flies will be subjected to PCR amplification with COI marker to determine the putative host species from Barcode of Life Data systems (BOLD) database. Following the comparative transcriptome analysis from the different species we will in vitro validate the presence and expression profiles of select set of putative olfaction related genes through qPCR. The functional role(s) of selected putative olfaction genes will be further investigated through RNAi gene silencing methodologies in conjunction with electro-antennogram and Wind-Tunnel techniques to establish the roles of these genes in modulating the physiology and behavior of the flies. Availability of comprehensive information on olfaction related genes, and their respective functional roles in modulating tsetse olfactory behavior will facilitate further R&D to exploit these genes to control tsetse fly vectors. This research will be done primarily in Kenya at International Centre of Insect Physiology Ecology and Kenya Agricultural Research Institute - Trypanosomiasis Research Center in collaboration with Paul Mireji with companion grant being Global Infectious Diseases (GID) D43 TW007391 (08/01/2008- 03/31/2013).